Download Overexpression of enhancer of zeste homolog 2

Carcinogenesis, 2015, Vol. 36, No. 11, 1333–1340
doi:10.1093/carcin/bgv137
Advance Access publication September 21, 2015
Original Manuscript
original manuscript
Overexpression of enhancer of zeste homolog 2 (EZH2)
characterizes an aggressive subset of prostate cancers
and predicts patient prognosis independently from
pre- and postoperatively assessed clinicopathological
parameters
Nathaniel Melling1,2,†, Erik Thomsen1,†, Maria Christina Tsourlakis1, Martina Kluth1,
Claudia Hube-Magg1, Sarah Minner1, Christina Koop1, Markus Graefen3, Hans Heinzer3,
Corinna Wittmer1, Guido Sauter1, Waldemar Wilczak1, Hartwig Huland3,
Ronald Simon1,*, Thorsten Schlomm3,4, Stefan Steurer1 and Till Krech1
Institute of Pathology, 2General, Visceral and Thoracic Surgery Department and Clinic, 3Martini-Clinic, Prostate Cancer
Center and 4Department of Urology, Section for translational Prostate Cancer Research, University Medical Center HamburgEppendorf 20246, Hamburg, Germany
1
*To whom correspondence should be addressed. Tel: +49 40 7410 57214; Fax: +49 40 7410 55997; Email: [email protected]
†
These authors contributed equally to this work.
Abstract
Enhancer of zeste homolog 2 (EZH2) plays an important role in tumor development and progression by interacting with
histone and nonhistone proteins. In the current study, we analyzed prevalence and prognostic impact of EZH2 in prostate
cancer. EZH2 expression was analyzed by immunohistochemistry on a tissue microarray containing more than 12 400
prostate cancer specimens. Results were compared to tumor phenotype, biochemical recurrence and molecular subtypes
defined by ERG status as well as genomic deletions of 3p, 5q, 6q and PTEN. EZH2 immunostaining was detectable in 56.6%
of 10 168 interpretable cancers and considered strong in 1.1%, moderate in 12.2% and weak in 43.3% of cases. High EZH2
expression was strongly associated with high Gleason grade (P < 0.0001), advanced pathological tumor stage (P < 0.0001),
positive nodal status (P < 0.0001), elevated preoperative PSA level (P = 0.0066), early PSA recurrence (P < 0.0001) and
increased cell proliferation P < 0.0001). High-level EZH2 staining was also associated with TMPRSS2:ERG rearrangement
and ERG expression in prostate cancers (P < 0.0001) and was linked to deletions of PTEN, 6q15, 5q21 and 3p13 (P < 0.0001
each) particularly in ERG-negative cancers. The prognostic impact of EZH2 was independent of established pre- and
postoperatively assessed clinicopathological parameters. EZH2 has strong prognostic impact in prostate cancer and might
contribute to the development of a fraction of genetically instable and particularly aggressive prostate cancers. EZH2
analysis might therefore be of clinical value for risk stratification of prostate cancer.
Introduction
Prostate cancer is the most prevalent cancer in men in Western
societies (1) Although most prostate cancers have a rather indolent
clinical course, this disease still represents the third most common cause of cancer-related death in men. A reliable distinction
between the indolent and the aggressive forms of the disease is
Received: July 8, 2015; Revised: September 9, 2015; Accepted: September 13, 2015
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected].
1333
1334 | Carcinogenesis, 2015, Vol. 36, No. 11
Abbreviations
EZH2
ERG
E26
PSA
PRC2
TMA
enhancer of zeste homolog 2
v-ets avian erythroblastosis virus
oncogene homolog
prostate-specific antigen
polycomb repressive complex 2
tissue microarray
highly desirable to improve therapy decisions. Despite recent
advances, the only established pretreatment prognostic parameters currently include Gleason grade and tumor extent on biopsies, preoperative prostate-specific antigen (PSA) and clinical stage.
Because these data are statistically powerful but not sufficient for
optimal individual treatment decisions, it can be hoped that a better understanding of disease biology will eventually lead to the
identification of clinically applicable molecular markers that enable a more reliable prediction of prostate cancer aggressiveness.
Enhancer of zeste homolog 2 (EZH2) is the catalytic part of the
Polycomb repressive complex 2 (PRC2), which regulates genes
involved in development and cell cycle progression (2). It catalyzes the trimethylation of Histone 3 on lysine 27 (H3K27me3)
and thus induces chromatin compaction and transcriptional
repression (2). This regulatory mechanism applies for the transcriptional control of various cancer related genes, including
for example the cell cycle regulator p16 and the cell adhesion
molecule E-Cadherin (3,4). In addition, EZH2 also interacts with
various nonhistone proteins such as STAT3, estrogen receptor
and beta catenin and thus indirectly serves as a transcriptional
activator of their target genes (5,6). It is thus not surprising that
overexpression of EZH2 has been implicated in initiation and
progression of various cancer entities such as breast cancer,
gastric cancer, bladder cancer and prostate cancer and has been
found to be associated with adverse clinicopathologic features
in all of these (7–10). Consequently, anti EZH2 therapies are currently under development (11–14). In prostate cancer studies on
64–259 primary tumors suggest that EZH2 overexpression may
be linked to adverse features of this disease and may represent a
potent predictor of patient prognosis (8,9,15–17).
These promising findings encouraged us to study the putative prognostic value of EZH2 in a large cohort including more
than 12 400 prostate cancers that have been assembled in a tissue microarray (TMA) format. The database attached to our TMA
contains pathological and clinical follow-up data, as well as data
on key molecular alterations, such as ERG fusion and genomic
deletion of FOXP1, 3p13, 5q21 and 6q15.
Materials and methods
Patients
Radical prostatectomy specimens were available from 12 427 patients,
undergoing surgery between 1992 and 2012 at the Department of Urology
and the Martini Clinics at the University Medical Center HamburgEppendorf. Follow-up data were available for a total of 12 344 patients with
a median follow-up of 36 months (range: 1–241 months; Table 1). PSA values were measured following surgery and PSA recurrence was defined as a
time point when postoperative PSA was at least 0.2 ng/ml and increasing at
subsequent measurements. All prostate specimens were analyzed according to a standard procedure, including a complete embedding of the entire
prostate for histological analysis (18). The TMA manufacturing process was
described earlier in detail (19). In short, one 0.6mm core was taken from a
representative tissue block from each patient. The tissues were distributed
among 27 TMA blocks, each containing 144–522 tumor samples. For internal
controls, each TMA block also contained various control tissues, including
normal prostate tissue. The usage of archived diagnostic left-over tissues
for manufacturing of TMAs and their analysis for research purposes as well
as patient data analysis has been approved by the local ethics committee
(Ethics commission Hamburg, WF-049/09 and PV3652). All work has been
carried out in compliance with the Helsinki Declaration. Usage of patient
data and routinely archived formalin fixed left-over patient tissue samples
for research purposes by the attending physician is approved by local laws
and does not require written consent (HmbKHG, §12,1). The molecular database attached to this TMA contained results on ERG expression in 10 711
(20), ERG break apart FISH analysis in 7122 (expanded from (21)) and deletion status of CHD1 (5q21) in 7932 (expanded from (22)), MAP3K7 (6q15) in
6069 (expanded from (23)), PTEN (10q23) in 6704 (expanded from (24)) and
FOXP1 (3p13) in 7081 (expanded from (25)) cancers. Immunohistochemical
data on Ki67-labelling index (Ki67-LI) and HDAC1 were available from 7010
cancers (expanded from (26)) and from 9744 cancers (27).
Immunohistochemistry
Freshly cut TMA sections were immunostained on 1 day and in one experiment. Slides were deparaffinized and exposed to heat-induced antigen
retrieval for 5 min in an autoclave at 121°C in pH 9 Tris-EDTA-Citrate buffer.
Primary antibody specific for EZH2 clone 6A10 (mouse monoclonal antibody, Abnova, Taipeh, Taiwan; cat#MAB9542; dilution 1:150) was applied at
37°C for 60 min. Bound antibody was then visualized using the EnVision
Kit (Dako, Glostrup, Denmark) according to the manufacturer´s directions.
Staining was predominantly nuclear and no staining was found in normal
tissue. EZH2 staining was typically found in either all (100%) or none (0%)
of the tumor cells in a given cancer spot. Staining intensity of all cases was
thus semiquantitatively assessed in four categories: negative, weak, moderate and strong. The percentage of positive tumor cells (typically 100%)
was not separately recorded.
Statistics
For statistical analysis, the JMP 10.0.2 software (SAS Institute Inc., NC) was
used. Contingency tables were calculated to study association between
EZH2 expression and clinicopathological variable, and the Chi-square
(Likelihood) test was used to find significant relationships. Kaplan–Meier
curves were generated for PSA recurrence free survival. The log-Rank
test was applied to test the significance of differences between stratified survival functions. Cox proportional hazards regression analysis was
performed to test the statistical independence and significance between
pathological, molecular, and clinical variables.
Results
Technical issues
A total of 10 168 (81.5%) of tumor samples were interpretable in
our TMA analysis. Reason for noninformative cases (2259 spots;
18.1%) included lack of tissue samples or absence of unequivocal cancer tissue in the TMA spot.
EZH2 immunohistochemistry
In normal prostatic glands, no EZH2 staining was observed. In cancers, EZH2 immunostaining was localized to the nucleus. Positive
staining was seen in 5755 of our 10 168 (56.6%) interpretable
tumors and was considered weak in 43.3%, moderate in 12.2% and
strong in 1.1% of cancers. Representative images of positive and
negative EZH2 immunostainings are given in Figure 1. Elevated
EZH2 expression was significantly linked to advanced pathological tumor stage (P < 0.0001), high Gleason grade (P < 0.0001),
lymph node metastases (P < 0.0001) and positive surgical margin
when all tumors were jointly analyzed (Table 2). A weaker but still
significant association was seen for high preoperative PSA levels
(P = 0.0066; Table 2). In subgroup analyses all of these associations were stronger in ERG negative than in ERG-positive cancers
(Supplementary Tables 1 and 2, available at Carcinogenesis Online).
Association with TMPRSS2:ERG fusion status and
ERG protein expression
To evaluate whether EZH2 expression is associated with ERG
status in prostate cancers, we used data from previous studies
N.Melling et al. | 1335
Table 1. Pathological and clinical data of the arrayed prostate cancers
No. of patients (%)
Follow-up (months)
n
Mean
Median
Age (years)
≤50
51–59
60–69
≥70
Pretreatment PSA (ng/ml)
<4
4–10
10–20
>20
pT stage (AJCC 2002)
pT2
pT3a
pT3b
pT4
Gleason grade
≤3 + 3
3 + 4
4 + 3
≥4 + 4
pN stage
pN0
pN+
Surgical margin
Negative
Positive
Study cohort on TMA (n = 12 427)
Biochemical relapse among categories
11 665 (93.9%)
48.9
36.4
2769 (23.7%)
—
—
334 (2.7%)
3061 (24.8%)
7188 (58.2%)
1761 (14.3%)
81 (24.3%)
705 (23%)
1610 (22.4%)
370 (21%)
1585 (12.9%)
7480 (60.9%)
2412 (19.6%)
812 (6.6%)
242 (15.3%)
1355 (18.1%)
737 (30.6%)
397 (48.9%)
8187 (66.2%)
2660 (21.5%)
1465 (11.8%)
63 (0.5%)
1095 (13.4%)
817 (30.7%)
796 (54.3%)
51 (81%)
2983 (24.1%)
6945 (56.2%)
1848 (15%)
584 (4.7%)
368 (12.3%)
1289 (18.6%)
788 (42.6%)
311 (53.3%)
6970 (91%)
693 (9%)
1636 (23.5%)
393 (56.7%)
9990 (81.9%)
2211 (18.1%)
1848 (18.5%)
853 (38.6%)
Numbers do not always add up to 12 427 in the different categories because of cases with missing data. Percentage in the column ‘Study cohort on TMA’ refers to the
fraction of samples across each category. Percentage in column ‘Biochemical relapse among categories’ refers to the fraction of samples with biochemical relapse
within each parameter in the different categories. AJCC, American Joint Committee on Cancer.
(expanded from (20,21)). Data on TMPRSS2:ERG fusion status
obtained by FISH were available from 5977 and by immunohistochemistry from 8943 tumors with evaluable EZH2 immunostaining. Data on both ERG FISH and IHC were available from
5744 cancers, and an identical result (ERG IHC positive and break
by FISH or ERG IHC negative and missing break by FISH) was
found in 5485 of 5744 (95.5%) cancers. EZH2 staining was more
frequent in TMPRSS2:ERG rearranged and ERG-positive prostate
cancers. Positive EZH2 immunostaining was seen in 65.2% (ERG
IHC) and 68.3% (ERG FISH) of ERG-positive cancers, and in 52.4
and 58.3% of cancers without ERG staining and ERG rearrangement, respectively (P < 0.0001 each; Figure 2). This difference
in overall EZH2 staining was largely due to more weak staining cases in ERG-positive cancers than in ERG-negative cancers,
while moderate and strong EZH2 staining showed comparable
frequencies in both subgroups (Figure 2).
Association to other key genomic deletions
Earlier studies had provided evidence for distinct molecular
subgroups of prostate cancers defined by TMPRSS2:ERG fusions
and several genomic deletions. Others and we had described
previously a strong link of PTEN and 3p13 deletions to ERG positivity and of 5q21 and 6q15 deletions to ERG negativity (22,23,25).
To examine, whether EZH2 expression might be particularly
associated with one of these genomic deletions, EZH2 data
were compared to preexisting findings on PTEN (10q23), FOXP1
(3p13), MAP3K7 (6q15) and CHD1 (5q21) deletions. Elevated EZH2
expression levels were strongly and consistently linked to all of
these deletions if all cancers were jointly analyzed, as well as in
the subset of ERG-negative cancers (P < 0.0001 each, Figure 3a
and b). These associations were clearly attenuated but still significant in ERG-positive cancers (Figure 3c).
Association to tumor cell proliferation (Ki67-LI)
EZH2 overexpression was significantly linked to increased cell
proliferation as measured by Ki67-LI in all cancers and in subgroup analyses by Gleason grade and ERG status (P < 0.0001
each). ERG negative and Gleason grade ≥4 + 4 cancers with strong
EZH2 expression displayed a particularly high proliferative fraction (P < 0.0001 each). Again this association was attenuated in
ERG-positive cancers. All comparisons with the Ki67-LI are summarized in Supplementary Table 3, available at Carcinogenesis
Online.
Association with PSA recurrence
Follow-up data were available from 9149 patients with interpretable EZH2 immunostaining on the TMA. Tumors with moderate or strong EZH2 immunostaining showed a significantly
shortened PSA recurrence-free interval if all cancers were
jointly analyzed and also in the subgroup analyses of ERGfusion negative and positive cancers (P < 0.0001, Figure 4a–c).
Because EZH2 activity has been suggested to depend on the
1336 | Carcinogenesis, 2015, Vol. 36, No. 11
Figure 1. Representative pictures of EZH2 immunostaining in prostate cancer with (a) negative, (b) weak, (c) moderate and (d) strong staining.
Table 2. Association between EZH2 immunostaining results and
prostate cancer phenotype in all cancers
(N)
Parameter
EZH2 (%)
Evaluable Negative Weak Moderate Strong P
All cancers 10 168
43.4
Tumor stage
pT2
6574
47.2
pT3a
2250
36.7
pT3b-pT4 1303
35.0
Gleason grade
≤3 + 3
2346
55.3
3 + 4
5717
41.8
4 + 3
1580
34.7
≥4 + 4
472
30.1
Lymph node metastasis
N0
5727
41.3
N+
594
32.0
Preoperative PSA level (ng/ml)
<4
1251
44.8
4–10
6052
43.6
10–20
2043
42.0
>20
713
41.2
Surgical margin
Negative 8045
44.8
Positive
1934
38.1
43.3
12.2
1.1
43.1
45.2
41.6
9.2
16.6
20.1
0.5
1.5
3.3
<0.0001
38.9
46.5
40.6
37.1
5.6
11.2
21.8
26.3
0.1
0.5
2.9
6.6
<0.0001
44.1
40.2
13.5
23.4
1.2
4.4
<0.0001
40.8
43.9
43.8
42.4
12.7
11.7
13.1
14.4
1.8
0.8
1.1
2.0
0.0066
43.1
43.8
11.2
16.1
0.8
2.0
<0.0001
with negative, weak and moderate HDAC1 expression (HDAC1
low). For the combined analysis, we further defined the EZH2/
HDAC1 status per tumor as follows: Tumors with low expression of both EZH2 and HDAC1 (both low), tumors with low
expression of one protein but high expression of the other
(one low/one high) and tumors with high expression of both
proteins (both high). This analysis revealed an increasingly
unfavorable outcome in patients with increasing levels of both
proteins (P < 0.0001, Figure 4d).
Multivariate analysis
activity of histone deacetylases (HDACs) (9), we performed an
additional analysis after combining EZH2 and HDAC1 immunostaining results, which we have previously analyzed on the
same TMA (27). In order to reduce data complexity we grouped
EZH2 into subsets with moderate or strong expression (EZH2
high) and those with negative or weak expression (EZH2 low).
Accordingly, we regrouped our previous HDAC1 data into subsets with strong HDAC1 expression (HDAC1 high) and those
Four different types of multivariate analyses were performed
evaluating the clinical relevance of EZH2 expression in different scenarios (Supplementary Table 4, available at Carcinogenesis
Online). Scenario 1 evaluated all postoperatively available
parameters including pathological tumor stage, pathological
lymph node status (pN), surgical margin status, preoperative
PSA value and pathological Gleason grade obtained after the
morphological evaluation of the entire resected prostate. In scenario 2, all postoperatively available parameters with exception
of nodal status were included. The rational for this approach
was that the indication and extent of lymph node dissection
is not standardized in the surgical therapy of prostate cancer
and that excluding pN in multivariate analysis can markedly
increase case numbers. Two additional scenarios had the purpose to model the preoperative situation as much as possible.
Scenario 3 included EZH2 expression, preoperative PSA, clinical tumor stage (cT stage) and Gleason grade obtained on the
prostatectomy specimen. Since postoperative determination
of a tumors Gleason grade is ‘better’ than the preoperatively
determined Gleason grade (subjected to sampling errors and
consequently under-grading in more than one third of cases
(28)), another multivariate analysis was added. In scenario 4, the
preoperative Gleason grade obtained on the original biopsy was
combined with preoperative PSA, cT stage and EZH2 expression. EZH2 expression proved to be an independent prognostic
N.Melling et al. | 1337
Figure 2. Association between EZH2 immunostaining results and the ERG status determined by IHC and FISH analysis. Breakage indicates rearrangement of the ERG
gene according to FISH analysis.
parameter in all scenarios when all tumors were analyzed
(P < 0.0001 each). These associations also held true in subgroup
analyses by ERG status, albeit attenuated in ERG-positive cancers (Supplementary Table 4, available at Carcinogenesis Online).
Discussion
The results of this study demonstrate that overexpression of
EZH2 protein is a strong and independent predictor of PSA
recurrence in prostate cancer, and that this effect is more
substantial in ERG negative than in ERG-positive carcinomas.
Detectable EZH2 expression was found in cancerous prostate epithelium but absent in normal epithelium in our study,
suggesting a role for increased EZH2 expression in prostate
cancer development. This finding is in agreement with an earlier study of Varambally et al. (9) reporting increasing EZH2
levels from normal epithelium to prostate intra-epithelial
neoplasia (PIN) and prostatic carcinoma. A total of 56.6% of
prostate cancers showed detectable EZH2 expression in our
study. Earlier studies analyzing TMAs with 64–259 primary
prostate cancers reported about 8–90% EZH2 cancers with relevant EZH2 staining. However, these studies employed various different antibodies and scoring criteria to distinguish
between lower and higher levels of EZH2 expression, hampering a direct comparison with our data (8,9,15–17,29,30). The
particularly high levels of EZH2 expression in high grade and
advanced prostate cancers suggest a substantial contribution of EZH2 expression to tumor progression. In line with
our findings, significant associations of EZH2 expression with
tumor stage, grade, nodal metastases, increased proliferation and unfavorable outcome have been suggested in several
earlier studies analyzing up to 292 prostate cancers for EZH2
expression (8,9,15,17).
Known reasons for a tumor-promoting role of EZH2 overexpression in prostate and other cancers include its documented
impact on the activity of pathways governing cellular functions
that are relevant for cancer such as cell growth, adhesion, motility and apoptosis (reviewed in (31)). Interestingly, EZH2 overexpression was strongly linked to the presence of ERG fusion in
our study, the most frequent molecular alteration in prostate
cancer. More than half of all prostate cancers, particularly those
of young patients, carry gene fusions linking the androgen-regulated TMPRSS2 gene with the transcription factor ERG (20,32).
These genomic rearrangements result in an androgen-driven
overexpression of ERG in affected cells (33) and, as a consequence, altered expression of more than 1600 genes in prostate
epithelial cells (34). The association of EZH2 overexpression and
TMPRSS2-ERG fusions is most probably caused by transactivation of the EZH2 promoter by ERG (35–37). Notably, it has been
suggested that ERG and EZH2 may synergistically promote prostate cancer progression based on their pivotal role in chromatin remodeling (37,38). Aberrations in chromatin remodeling
proteins are often found in prostate and other cancers. These
include alterations of histone modifiers, such as EZH2, and chromatin remodeling complexes that move, eject or restructure
nucleosomes (39–41). ERG has been shown to alter chromatin
structure by opening cryptic transcription factor binding sites
(38).
Next to TMPRSS2-ERG fusions, chromosomal deletions represent the second most frequent type of genomic aberrations
occurring at frequencies up to 40% in prostate cancer (42,43).
In particular, deletions of PTEN (20%), 6q (20%), 5q (10%) and 3p
(10%) belong to the most prevalent genomic alterations in this
disease, which are strongly linked to either positive (PTEN, 3p) or
negative ERG status (6q, 5q) and are associated with poor patient
prognosis (22–25). Remarkably, all of these genomic deletions,
some of which are inversely linked to each other, were associated with high levels of EZH2 expression, suggesting a role for
EZH2 overexpression in the induction of chromosomal instability. In fact, it is known that perturbations of chromatin structure
caused by alterations of polycomb group proteins such as EZH2
can result in genomic instability due to transcriptional deregulation of genes involved in maintenance of genomic integrity (44)
For example EZH2 overexpression in breast cancer cells induces
extra centrosomes and chromosomal instability by inhibiting
the cell cycle checkpoint protein BRCA1 (45) and by impairing
formation of RAD51 repair foci at sites of DNA strand breaks (46).
Irrespective of the mechanisms by which EZH2 provokes
tumor aggressiveness the data from our study demonstrate,
that EZH2 expression is one of the strongest prognostic features in prostate cancer described as to yet. Even though the
relationship with tumor phenotype and prognosis was somewhat less impressive in ERG-positive than in ERG-negative
tumors, EZH2 was a strong and independent prognostic feature
in both subgroups, which makes it a promising candidate for a
routine application. This is all the more true as EZH2 predicted
1338 | Carcinogenesis, 2015, Vol. 36, No. 11
Figure 3. Association between positive EZH2 immunostaining results and deletions of PTEN, 5q21 (CHD1), 6q15 (MAP3K7) and 3p13 (FOXP1) in (a) all cancers as well as
the subsets of (b) ERG-negative and (c) ERG-positive cancers according to ERG-IHC analysis.
prognosis irrespective of whether pre- or postoperative established prognostic features were included in multivariate analysis. It is also noteworthy, that our method of analyzing EZH2
in minute tissue cores largely mimics the analysis of core needle biopsies that would be available in a preoperative scenario.
A clinical benefit of EZH2 measurement in biopsies is also supported by two studies analyzing EZH2 expression by IHC in core
needle biopsies from 72 and 209 prostate cancer patients and
reporting significant associations of EZH2 expression with clinical outcome (16,30).
EZH2 has gained substantial interest as a target for epigenetic anticancer therapy. Recent preclinical studies described
significant effects of EZH2 in experimental systems derived
from cancers of the lung (12), ovary (11), as well as in melanomas (14) and hematological malignancies (13). Clinical
phase I/II studies using the EZH2 inhibitor E7438 have recently
been initiated in diffuse large B-cell lymphoma patients
(NCT01897571). Of note, it has been suggested that EZH2mediated gene silencing may depend on the activity of histone
deacetylases (HDACs) (9)—another promising therapy target in
prostate cancer (47,48)—and that a combinatorial epigenetic
therapy targeting both EZH2 and HDACs may enhance the antitumor activity in leukemia (49). We have earlier demonstrated a
strong prognostic role of HDAC1 expression in prostate cancer
(27). In the context of combining treatment targets, it is interesting that our combined analysis of HDAC1 and EZH2 in prostate cancer revealed an increasingly unfavorable outcome in
patients with increasing levels of both proteins. Simultaneous
EZH2 and HDAC1 targeting may, thus, be instrumental in prostate cancer.
N.Melling et al. | 1339
Figure 4. Association between EZH2 expression and biochemical recurrence (BCR) in (a) all cancers, (b) ERG fusion negative cancers, (c) ERG fusion positive cancers and
(d) combined impact of EZH2 and HDAC1 expression on BCR in all cancers.
Supplementary material
Supplementary Tables 1–4 can be found at http://carcin.oxfordjournals.org/
Funding
Project CancerTelSys (grant number 01ZX1302) in the E:med
program of the German Federal Ministry of Education and
Research (BMBF).
Acknowledgements
We thank Julia Schumann, Sünje Seekamp and Inge Brandt for
excellent technical assistance. N.M., E.T., C.H.M., G.S., R.S. and
T.K. conceived and designed the study, analyzed the data and
drafted the manuscript. C.B. and D.M. performed most of the key
immunohistochemical analyses. G.S. and R.S. were involved in
the original conception of the study. M.C.T., M.K., N.M., C.H.M. and
S.M. provided data. N.M., E.T., M.C.T., S.M., C.K., C.W., G.S., S.S.,
W.W. and T.K. participated in tissue processing, pathological diagnosis and immunohistochemical analysis. C.K., M.G., H.H., H.H.,
G.S., R.S. and T.S. provided materials, clinical follow-up data and
technical assistance. All authors have read and approved the manuscript. N.M. and E.T. analyzed all IHC experiments. M.T., S.M., S.S.,
C.W. and C.K. performed histological tissue examination, selected
tumors, and supervised TMA manufacturing. C.H.M. and M.K. performed statistical analysis. W.W., G.S., R.S., T.S. and T.K. interpreted
all data and wrote the manuscript. H.H., H.H. and M.G. supported
clinical data and all authors were involved in writing the paper
and had final approval of the submitted version.
Conflict of Interest Statement: None declared.
References
1. Torre, L.A. et al. (2015) Global cancer statistics, 2012. CA Cancer J. Clin.,
65, 87–108.
2. Sauvageau, M. et al. (2010) Polycomb group proteins: multi-faceted
regulators of somatic stem cells and cancer. Cell Stem Cell, 7, 299–313.
3. Cao, Q. et al. (2008) Repression of E-cadherin by the polycomb group
protein EZH2 in cancer. Oncogene, 27, 7274–7284.
4. Kotake, Y. et al. (2007) pRB family proteins are required for H3K27
trimethylation and Polycomb repression complexes binding to and
silencing p16INK4alpha tumor suppressor gene. Genes Dev., 21, 49–54.
5. Kim, E. et al. (2013) Phosphorylation of EZH2 activates STAT3 signaling
via STAT3 methylation and promotes tumorigenicity of glioblastoma
stem-like cells. Cancer Cell, 23, 839–852.
6. Shi, B. et al. (2007) Integration of estrogen and Wnt signaling circuits by
the polycomb group protein EZH2 in breast cancer cells. Mol. Cell. Biol.,
27, 5105–5119.
7. Kleer, C.G. et al. (2003) EZH2 is a marker of aggressive breast cancer
and promotes neoplastic transformation of breast epithelial cells. Proc.
Natl. Acad. Sci. USA, 100, 11606–11611.
8. Laitinen, S. et al. (2008) EZH2, Ki-67 and MCM7 are prognostic markers
in prostatectomy treated patients. Int. J. Cancer, 122, 595–602.
9. Varambally, S. et al. (2002) The polycomb group protein EZH2 is involved
in progression of prostate cancer. Nature, 419, 624–629.
10.Arisan, S. et al. (2005) Increased expression of EZH2, a polycomb group
protein, in bladder carcinoma. Urol. Int., 75, 252–257.
11.Bitler, B.G. et al. (2015) Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated cancers. Nat. Med., 21, 231–
238.
12.Fillmore, C.M. et al. (2015) EZH2 inhibition sensitizes BRG1 and EGFR
mutant lung tumours to TopoII inhibitors. Nature, 520, 239–242.
13.McCabe, M.T. et al. (2012) EZH2 inhibition as a therapeutic strategy for
lymphoma with EZH2-activating mutations. Nature, 492, 108–112.
14.Zingg, D. et al. (2015) The epigenetic modifier EZH2 controls melanoma
growth and metastasis through silencing of distinct tumour suppressors. Nat. Commun., 6, 6051.
15.Rhodes, D.R. et al. (2003) Multiplex biomarker approach for determining risk of prostate-specific antigen-defined recurrence of prostate
cancer. J. Natl. Cancer Inst., 95, 661–668.
16.Tolonen, T.T. et al. (2011) Histopathological variables and biomarkers
enhancer of zeste homologue 2, Ki-67 and minichromosome maintenance protein 7 as prognosticators in primarily endocrine-treated
prostate cancer. BJU Int., 108, 1430–1438.
17.Bachmann, I.M. et al. (2006) EZH2 expression is associated with high
proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast. J. Clin.
Oncol., 24, 268–273.
1340 | Carcinogenesis, 2015, Vol. 36, No. 11
18.Schlomm, T. et al. (2008) Clinical significance of p53 alterations in surgically treated prostate cancers. Mod. Pathol., 21, 1371–1378.
19.Kononen, J. et al. (1998) Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat. Med., 4, 844–847.
20.Weischenfeldt, J. et al. (2013) Integrative genomic analyses reveal an
androgen-driven somatic alteration landscape in early-onset prostate
cancer. Cancer Cell, 23, 159–170.
21.Minner, S. et al. (2011) ERG status is unrelated to PSA recurrence in
radically operated prostate cancer in the absence of antihormonal
therapy. Clin. Cancer Res., 17, 5878–5888.
22.Burkhardt, L. et al. (2013) CHD1 is a 5q21 tumor suppressor required for
ERG rearrangement in prostate cancer. Cancer Res., 73, 2795–2805.
23.Kluth, M. et al. (2013) Genomic deletion of MAP3K7 at 6q12-22 is associated with early PSA recurrence in prostate cancer and absence of
TMPRSS2:ERG fusions. Mod. Pathol., 26, 975–983.
24.Krohn, A. et al. (2012) Genomic deletion of PTEN is associated with
tumor progression and early PSA recurrence in ERG fusion-positive
and fusion-negative prostate cancer. Am. J. Pathol., 181, 401–412.
25.Krohn, A. et al. (2013) Recurrent deletion of 3p13 targets multiple
tumour suppressor genes and defines a distinct subgroup of aggressive ERG fusion-positive prostate cancers. J. Pathol., 231, 130–141.
26.Minner, S. et al. (2010) Low level HER2 overexpression is associated
with rapid tumor cell proliferation and poor prognosis in prostate cancer. Clin. Cancer Res., 16, 1553–1560.
27.Burdelski, C. et al. (2015) HDAC1 overexpression independently predicts biochemical recurrence and is associated with rapid tumor cell
proliferation and genomic instability in prostate cancer. Exp. Mol.
Pathol., 98, 419–426.
28.Epstein, J.I. et al. (2012) Upgrading and downgrading of prostate cancer
from biopsy to radical prostatectomy: incidence and predictive factors
using the modified Gleason grading system and factoring in tertiary
grades. Eur. Urol., 61, 1019–1024.
29.Saramäki, O.R. et al. (2006) The gene for polycomb group protein
enhancer of zeste homolog 2 (EZH2) is amplified in late-stage prostate
cancer. Genes. Chromosomes Cancer, 45, 639–645.
30.Wolters, T. et al. (2010) The value of EZH2, p27(kip1), BMI-1 and MIB-1
on biopsy specimens with low-risk prostate cancer in selecting men
with significant prostate cancer at prostatectomy. BJU Int., 106, 280–
286.
31.Yamaguchi, H. et al. (2014) Regulation and Role of EZH2 in Cancer. Cancer Res. Treat., 46, 209–222.
32.Tomlins, S.A. et al. (2005) Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science, 310, 644–648.
33.Clark, J.P. et al. (2009) ETS gene fusions in prostate cancer. Nat. Rev.
Urol., 6, 429–439.
34.Brase, J.C. et al. (2011) TMPRSS2-ERG -specific transcriptional modulation is associated with prostate cancer biomarkers and TGF-β signaling. BMC Cancer, 11, 507.
35.Börno, S.T. et al. (2012) Genome-wide DNA methylation events in
TMPRSS2-ERG fusion-negative prostate cancers implicate an EZH2dependent mechanism with miR-26a hypermethylation. Cancer Discov., 2, 1024–1035.
36.Kunderfranco, P. et al. (2010) ETS transcription factors control transcription of EZH2 and epigenetic silencing of the tumor suppressor
gene Nkx3.1 in prostate cancer. PLoS One, 5, e10547.
37.Yu, J. et al. (2010) An integrated network of androgen receptor, polycomb, and TMPRSS2-ERG gene fusions in prostate cancer progression.
Cancer Cell, 17, 443–454.
38. Cai, C. et al. (2013) ERG induces androgen receptor-mediated regulation
of SOX9 in prostate cancer. J. Clin. Invest., 123, 1109–1122.
39. Chi, P. et al. (2010) Covalent histone modifications–miswritten, misinterpreted and mis-erased in human cancers. Nat. Rev. Cancer, 10, 457–469.
40.Glozak, M.A. et al. (2007) Histone deacetylases and cancer. Oncogene,
26, 5420–5432.
41.Hargreaves, D.C. et al. (2011) ATP-dependent chromatin remodeling:
genetics, genomics and mechanisms. Cell Res., 21, 396–420.
42.Sun, J. et al. (2007) DNA copy number alterations in prostate cancers: a
combined analysis of published CGH studies. Prostate, 67, 692–700.
43. Taylor, B.S. et al. (2010) Integrative genomic profiling of human prostate
cancer. Cancer Cell, 18, 11–22.
44.Sparmann, A. et al. (2006) Polycomb silencers control cell fate, development and cancer. Nat. Rev. Cancer, 6, 846–856.
45.Gonzalez, M.E. et al. (2011) Histone methyltransferase EZH2 induces
Akt-dependent genomic instability and BRCA1 inhibition in breast
cancer. Cancer Res., 71, 2360–2370.
46.Zeidler, M. et al. (2005) The Polycomb group protein EZH2 impairs DNA
repair in breast epithelial cells. Neoplasia, 7, 1011–1019.
47.Butler, L.M. et al. (2000) Suberoylanilide hydroxamic acid, an inhibitor
of histone deacetylase, suppresses the growth of prostate cancer cells
in vitro and in vivo. Cancer Res., 60, 5165–5170.
48.Rathkopf, D. et al. (2010) A phase I study of oral panobinostat alone and
in combination with docetaxel in patients with castration-resistant
prostate cancer. Cancer Chemother. Pharmacol., 66, 181–189.
49.Fiskus, W. et al. (2006) Histone deacetylase inhibitors deplete enhancer
of zeste 2 and associated polycomb repressive complex 2 proteins in
human acute leukemia cells. Mol. Cancer Ther., 5, 3096–3104.